In response to different stresses, eukaryotic cells dramatically reduce protein synthesis by phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF- 2). Recently, we identified a new eIF-2alpha kinase from rat pancreas. The new protein kinase designated pancreatic eIF-2alpha kinase, PEK, is an ER transmembrane protein that is activated in response to ER stresses that impair protein folding in this organelle. PEK is expressed in all tissues examined, with highest levels in secretory tissues. PEK sequences in the ER lumen are proposed to sense ER stress, eliciting a conformation change in PEK that stimulates phosphorylation of eIF-2alpha. Reduced protein synthesis provides the cell an opportunity to remedy protein misfolding prior to introducing newly synthesized proteins into the secretory pathway. Two fundamental questions will be addressed in this proposal. First, we will address the mechanisms regulating PEK in response to ER stress. We consider two hypotheses for the regulation of PEK activity. First, the stressed ER may directly modify PEK or an associated cofactor. For example, a change in the oxidizing conditions of the ER may alter the disulfide structure of PEK or an associated cofactor, leading to an active protein conformation and enhanced autophosphorylation. The second hypothesis we consider is that ER stress is monitored by an ER resident protein, such as the chaperone GRP78/BiP, that associates with the amino terminus of PEK, maintaining it in an inactive conformation. During ER stress, GRP78 may bind to unfolded proteins that accumulates in the lumen of the ER, freeing PEK to oligomerize and trans- autophosphorylate. We also explore the possibility that proteins known to regulate the related eIF-2alpha kinase, PKR, that is involved in an antiviral defense pathway, also controls PEK function. In this fashion there would be overlapping regulatory mechanisms between these two translational control systems. The second question we will address concerns whether PEK phosphorylation of eIF-2alpha uniformly reduces protein synthesis. During ER stress there is increased transcriptional expression of many ER proteins that serve to remedy stress- mediated protein misfolding. How are these proteins expressed at elevated levels during a general reduction in translation? To answer these questions, we propose four specific aims: 1) Characterization of PEK sequences involved in the activation of eIF-2alpha kinase activity during ER stress; 2) Identification and characterization of regulatory proteins interacting with PEK; 3) Characterization of PEK control of protein synthesis in response to ER stress; and 4) Characterize the control of PEK activity by known PKR regulatory proteins. Together, these studies will further our understanding of the mechanisms regulating general and gene-specific protein synthesis during ER stress.